Maize metallothionein 2 promoter and methods of use

ABSTRACT

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for a promoter for the gene encoding metallothionein. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant or plant cell with a nucleotide sequence operably linked to one of the promoters of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/531,793 filed on Dec. 22, 2003 and U.S. Provisional Application No.60/532,180, filed on Dec. 23, 2003, both of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of an operably linked promoter that is functionalwithin the plant host. Choice of the promoter sequence will determinewhen and where within the organism the heterologous DNA sequence isexpressed. Where expression in specific tissues or organs is desired,tissue-preferred promoters may be used. Where gene expression inresponse to a stimulus is desired, inducible promoters are theregulatory element of choice. In contrast, where continuous expressionis desired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in the expressionconstructs of transformation vectors to bring about varying levels ofexpression of heterologous nucleotide sequences in a transgenic plant.

Frequently it is desirable to express a DNA sequence in particulartissues or organs of a plant. For example, increased resistance of aplant to infection by soil- and air-borne pathogens might beaccomplished by genetic manipulation of the plant's genome to comprise atissue-preferred promoter operably linked to a heterologouspathogen-resistance gene such that pathogen-resistance proteins areproduced in the desired plant tissue.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a tissue-preferred promoter operably linked to anantisense nucleotide sequence, such that expression of the antisensesequence produces an RNA transcript that interferes with translation ofthe mRNA of the native DNA sequence.

Thus far, the regulation of gene expression in plant roots has not beenadequately studied despite the importance of the root to plantdevelopment. To some degree this is attributable to a lack of readilyavailable, root-specific biochemical functions whose genes may becloned, studied, and manipulated. Genetically altering plants throughthe use of genetic engineering techniques and thus producing a plantwith useful traits requires the availability of a variety of promoters.An accumulation of promoters would enable the investigator to designrecombinant DNA molecules that are capable of being expressed at desiredlevels and cellular locales. Therefore, a collection of tissue-preferredpromoters would allow for a new trait to be expressed in the desiredtissue. Several genes have been described by Takahashi et al. (1991)Plant J 1: 327-332; Takahashi et al. (1990) Proc. Natl. Acad. Sci. USA87: 8013-8016; Hertig et al. (1991) Plant Mol. Biol. 16: 171-174; Xu etal. (1995) Plant Mol. Biol. 27: 237-248; Capone et al. (1994) Plant Mol.Biol. 25: 681-691; Masuda et al. (1999) Plant Cell Physiol. 40(11):1177-81; Luschnig et al. (1998) Genes Dev. 12(14): 2175-87; Goddemeieret al. (1998) Plant Mol. Biol. 36(5): 799-802; and Yamamoto et al.(1991) Plant Cell. 3(4): 371-82 that express preferentially in plantroot tissues.

Metallothioneins (MT's) are proteins or polypeptides that bind andsequester ionic forms of certain metals in plant and animal tissues.Examples of such metals include copper, zinc, cadmium, mercury, gold,silver, cobalt, nickel and bismuth. The specific metals sequestered byMT's vary for the structurally distinct proteins/polypeptides occurringin different organisms. Plants appear to contain a diversity ofmetal-binding MT's with the potential to perform distinct roles in themetabolism of different metal ions. In plants, MT's are suggested tohave roles in metal accumulation, metal intoxication, and embryogenesis(Thomas et al. (2003) Biotechnol. Prog. 19: 273-280; Dong and Dunstan(1996) Planta 199: 459-466; Kawashima et al. (1992) Eur. J. Biochem.209: 971-976).

Typically, MT's and MT-like proteins are Cys-rich proteins that arecharacterized by the presence of Cys-Xaa-Cys motifs suggesting thecapability of binding metal ions. Further categories of MT-like proteinshave been proposed on the basis of the predicted locations of Cysresidues and have been designated types 1 and 2. In type 1 there areexclusively Cys-Xaa-Cys motifs, whereas in type 2 there is a Cys-Cys anda Cys-Xaa-Xaa-Cys pair within the N-terminal domain. The type 1 motifhas been implicated in the binding and sequestration of copper. (Murphyet al. (1997) Plant Physiol. 113: 1293-1301 and Carr et al. (2002) J.Biol. Chem. 277: 31237-31242) Several metallothionein-like plant geneshave been isolated, including those from pea, maize, barley, Mimulus(monkeyflower), soybean, castor bean and Arabidopsis. See Robinson etal. (1993) Biochem J 295: 1-10. Sequences expressed in roots have beenreported to be isolated from pea, as described in Evans et al. (1990)FEBS Lett 262: 29-32. A maize root MT gene has been described in U.S.Pat. No. 5,466,785; though this sequence is also expressed in leaves,pith and seed, as described in de Framond (1991) FEBS Lett 290: 103-106.

Thus, isolation and characterization of tissue-preferred, particularlyroot-preferred, promoters that can serve as regulatory regions forexpression of heterologous nucleotide sequences of interest in atissue-preferred manner are needed for genetic manipulation of plants.

SUMMARY OF THE INVENTION

Compositions and methods for regulating expression of a heterologousnucleotide sequence of interest in a plant or plant cell are provided.Compositions comprise novel nucleotide sequences for promoters thatinitiate transcription. Embodiments of the invention comprise thenucleotide sequence set forth in SEQ ID NO: 1 or a complement thereof,the nucleotide sequence comprising the plant promoter sequence of theplasmid deposited as Patent Deposit No. NRRL B-30793 or a complementthereof, a nucleotide sequence comprising at least 20 contiguousnucleotides of SEQ ID NO: 1, wherein said sequence initiatestranscription in a plant cell, and a nucleotide sequence comprising asequence having at least 85% sequence identity to the sequence set forthin SEQ ID NO:1, wherein said sequence initiates transcription in theplant cell.

A method for expressing a heterologous nucleotide sequence in a plant orplant cell is provided. The method comprises introducing into a plant ora plant cell an expression cassette comprising a heterologous nucleotidesequence of interest operably linked to one of the promoters of thepresent invention. In this manner, the promoter sequences are useful forcontrolling the expression of the operably linked heterologousnucleotide sequence. In specific methods, the heterologous nucleotidesequence of interest is expressed in a root-preferred manner.

Further provided is a method for expressing a nucleotide sequence ofinterest in a root-preferred manner in a plant. The method comprisesintroducing into a plant cell an expression cassette comprising apromoter of the invention operably linked to a heterologous nucleotidesequence of interest.

Expression of the nucleotide sequence of interest can provide formodification of the phenotype of the plant. Such modification includesmodulating the production of an endogenous product, as to amount,relative distribution, or the like, or production of an exogenousexpression product to provide for a novel function or product in theplant. In specific methods and compositions, the heterologous nucleotidesequence of interest comprises a gene product that confers herbicideresistance, pathogen resistance, insect resistance, and/or alteredtolerance to salt, cold, or drought.

Expression cassettes comprising the promoter sequences of the inventionoperably linked to a heterologous nucleotide sequence of interest areprovided. Additionally provided are transformed plant cells, planttissues, seeds, and plants.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods drawn to plantpromoters and methods of their use. The compositions comprise nucleotidesequences for the promoter region of the metallothionein (MT) gene. Thecompositions further comprise DNA constructs comprising a nucleotidesequence for the promoter region of the metallothionein 1 (MT2) geneoperably linked to a heterologous nucleotide sequence of interest. Inparticular, the present invention provides for isolated nucleic acidmolecules comprising the nucleotide sequence set forth in SEQ ID NO: 1,and the plant promoter sequence deposited in bacterial hosts as PatentDeposit No. NRRL B-30793, on Dec. 1, 2004, and fragments, variants, andcomplements thereof.

Plasmids containing the plant promoter nucleotide sequences of theinvention were deposited on Dec. 1, 2004 with the Patent Depository ofthe Agricultural Research Service Culture Collection of the NationalCenter for Agricultural Utilization Research, at 1815 N. UniversityStreet, Peoria, Ill., 61604, and assigned Patent Deposit No. NRRLB-30793. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112. The deposit will irrevocablyand without restriction or condition be available to the public uponissuance of a patent. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

The MT2 promoter sequences of the present invention include nucleotideconstructs that allow initiation of transcription in a plant. Inspecific embodiments, the MT2 promoter sequence allows initiation oftranscription in a tissue-preferred, more particularly in aroot-preferred manner. Such constructs of the invention compriseregulated transcription initiation regions associated with plantdevelopmental regulation. Thus, the compositions of the presentinvention include DNA constructs comprising a nucleotide sequence ofinterest operably linked to a plant promoter, particularlyroot-preferred promoter sequences for the MT2 gene, more particularly amaize MT2 promoter sequence. The sequence for the maize MT2 promoterregion is set forth in SEQ ID NO: 1.

Compositions of the invention include the nucleotide sequences for thenative MT2 promoter and fragments and variants thereof. The promotersequences of the invention are useful for expressing sequences. Inspecific embodiments, the promoter sequences of the invention are usefulfor expressing sequences of interest in a tissue-preferred, particularlya root-preferred manner. The nucleotide sequences of the invention alsofind use in the construction of expression vectors for subsequentexpression of a heterologous nucleotide sequence in a plant of interestor as probes for the isolation of other MT2-like promoters.

Related metallothionein promoter sequences are disclosed in U.S.application Ser. No. 09/520,268 and in U.S. Provisional Application No.60/531,793 filed Dec. 22, 2003, the disclosures of which are hereinincorporated by reference. In particular, the present invention providesfor isolated DNA constructs comprising the MT2 promoter nucleotidesequence set forth in SEQ ID NO: 1 operably to a nucleotide sequence ofinterest.

The invention encompasses isolated or substantially purified nucleicacid compositions. An “isolated” or “purified” nucleic acid molecule orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acid isfree of sequences (optimally protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. The MT2 promoter sequences of the inventionmay be isolated from the 5′ untranslated region flanking theirrespective transcription initiation sites.

Fragments and variants of the disclosed promoter nucleotide sequencesare also encompassed by the present invention. In particular, fragmentsand variants of the MT2 promoter sequence of SEQ ID NO: 1 may be used inthe DNA constructs of the invention. As used herein, the term “fragment”refers to a portion of the nucleic acid sequence. Fragments of an MT2promoter sequence may retain the biological activity of initiatingtranscription, more particularly driving transcription in aroot-preferred manner. Alternatively, fragments of a nucleotide sequencethat are useful as hybridization probes may not necessarily retainbiological activity. Fragments of a nucleotide sequence for the promoterregion of the MT2 gene may range from at least about 20 nucleotides,about 50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence of the invention for the promoter region of thegene.

A biologically active portion of an MT2 promoter can be prepared byisolating a portion of the MT2 promoter sequence of the invention, andassessing the promoter activity of the portion. Nucleic acid moleculesthat are fragments of an MT2 promoter nucleotide sequence comprise atleast about 16, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300 nucleotides, or upto the number of nucleotides present in a full-length MT2 promotersequence disclosed herein (for example, 1347 nucleotides for SEQ ID NO:1).

As used herein, the term “variants” means substantially similarsequences. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, suchas, for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein.

For nucleotide sequences, a variant comprises a deletion and/or additionof one or more nucleotides at one or more internal sites within thenative polynucleotide and/or a substitution of one or more nucleotidesat one or more sites in the native polynucleotide. As used herein, a“native” nucleotide sequence comprises a naturally occurring nucleotidesequence. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a nucleotide sequence of the invention may differ fromthat sequence by as few as 1-15 nucleic acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleic acidresidue.

Variant nucleotide sequences also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. With sucha procedure, one or more different MT2 nucleotide sequences for thepromoter can be manipulated to create a new MT2 promoter. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri etal. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol.Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Pat. Nos.5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire MT2 sequencesset forth herein or to fragments thereof are encompassed by the presentinvention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from genomic DNAextracted from any plant of interest. Methods for designing PCR primersand PCR cloning are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafterSambrook. See also Innis et al., eds. (1990) PCR Protocols: A Guide toMethods and Applications (Academic Press, New York); Innis and Gelfand,eds. (1995) PCR Strategies (Academic Press, New York); and Innis andGelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).Known methods of PCR include, but are not limited to, methods usingpaired primers, nested primers, single specific primers, degenerateprimers, gene-specific primers, vector-specific primers,partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments from a chosen organism. The hybridization probes may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the MT2 promoter sequencesof the invention. Methods for preparation of probes for hybridizationand for construction of genomic libraries are generally known in the artand are disclosed in Sambrook.

For example, the entire MT2 promoter sequence disclosed herein, or oneor more portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding MT2 promoter sequences and messenger RNAs.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among MT2 promoter sequencesand are at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingMT2 promoter sequences from a chosen plant by PCR. This technique may beused to isolate additional coding sequences from a desired organism, oras a diagnostic assay to determine the presence of coding sequences inan organism. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Cloning: A Laboratory Manual (2^(nd) ed, ColdSpring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. The terms “stringent conditions” or “stringent hybridizationconditions” are intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for a duration of atleast 30 minutes. Duration of hybridization is generally less than about24 hours, usually about 4 to about 12 hours. The duration of the washtime will be at least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) (thermal melting point)can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138: 267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See also Sambrook.

Thus, isolated sequences that have root-preferred promoter activity andwhich hybridize under stringent conditions to the MT2 promoter sequencesdisclosed herein, or to fragments thereof, are encompassed by thepresent invention.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al.(1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et al (1990) J.Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. An“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the GCG Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89: 10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, optimally at least 80%, more optimally at least 90%,and most optimally at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species include corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), ahnond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Heterologous coding sequences expressed by the MT2 promoters of theinvention may be used for varying the phenotype of a plant. Variouschanges in phenotype are of interest including modifying expression of agene in a plant root, altering a plant's pathogen or insect defensemechanism, increasing the plants tolerance to herbicides in a plant,altering root development to respond to environmental stress, modulatingthe plant's response to salt, temperature (hot and cold), drought, andthe like. These results can be achieved by the expression of aheterologous nucleotide sequence of interest comprising an appropriategene product. In specific embodiments, the heterologous nucleotidesequence of interest is an endogenous plant sequence whose expressionlevel is increased in the plant or plant part. Alternatively, theresults can be achieved by providing for a reduction of expression ofone or more endogenous gene products, particularly enzymes,transporters, or cofactors, or by affecting nutrient uptake in theplant. These changes result in a change in phenotype of the transformedplant.

General categories of nucleotide sequences of interest for the presentinvention include, for example, those genes involved in information,such as zinc fingers, those involved in communication, such as kinases,and those involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes, for example, include genes encodingimportant traits for agronomics, insect resistance, disease resistance,herbicide resistance, and environmental stress resistance (alteredtolerance to cold, salt, drought, etc). It is recognized that any geneof interest can be operably linked to the promoter of the invention andexpressed in the plant.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European corn borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as those which detoxify fumonisin (U.S. Pat. No. 5,792,931);avirulence (avr) and disease resistance (R) genes (Jones et al. (1994)Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinoset al. (1994) Cell 78: 1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene),glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360) or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Glyphosate resistance is imparted by mutant 5-enolpyruvl-3-phosphikimatesynthase (EPSP) and aroA genes. See, for example, U.S. Pat. No.4,940,835 to Shah et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes.See also U.S. Pat. Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and internationalpublications WO 97/04103; WO 97/04114; WO 00/66746; WO 01/66704; WO00/66747 and WO 00/66748, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene that encodes a glyphosate oxido-reductase enzyme asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, whichare incorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.patent application Ser. Nos. 10/004,357; and 10/427,692.

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like.

Examples of other applicable genes and their associated phenotypeinclude the gene which encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that promote yield improvement; and genes thatprovide for resistance to stress, such as cold, dehydration resultingfrom drought, heat and salinity, toxic metal or trace elements, or thelike.

As noted, the heterologous nucleotide sequence operably linked to theMT2 promoters disclosed herein may be an antisense sequence for atargeted gene. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant root.

“RNAi” refers to a series of related techniques to reduce the expressionof genes (See for example U.S. Pat. No. 6,506,559). Older techniquesreferred to by other names are now thought to rely on the samemechanism, but are given different names in the literature. Theseinclude “antisense inhibition,” the production of antisense RNAtranscripts capable of suppressing the expression of the target protein,and “co-suppression” or “sense-suppression,” which refer to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020, incorporated herein by reference). Suchtechniques rely on the use of constructs resulting in the accumulationof double stranded RNA with one strand complementary to the target geneto be silenced. The MT2 promoters of the embodiments may be used todrive expression of constructs that will result in RNA interferenceincluding microRNAs and siRNAs.

As used herein, the terms “promoter” or “transcriptional initiationregion” mean a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter may additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter regions disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe 5′ untranslated region upstream from the particular promoter regionsidentified herein. Additionally, chimeric promoters may be provided.Such chimeras include portions of the promoter sequence fused tofragments and/or variants of heterologous transcriptional regulatoryregions. Thus, the promoter regions disclosed herein can compriseupstream regulatory elements such as, those responsible for tissue andtemporal expression of the coding sequence, enhancers and the like. Inthe same manner, the promoter elements, which enable expression in thedesired tissue such as the root, can be identified, isolated and usedwith other core promoters to confer root-preferred expression. In thisaspect of the invention, “core promoter” is intended to mean a promoterwithout promoter elements.

In the context of this disclosure, the term “regulatory element” alsorefers to a sequence of DNA, usually, but not always, upstream (5′) tothe coding sequence of a structural gene, which includes sequences whichcontrol the expression of the coding region by providing the recognitionfor RNA polymerase and/or other factors required for transcription tostart at a particular site. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.A promoter element comprises a core promoter element, responsible forthe initiation of transcription, as well as other regulatory elements(as discussed elsewhere in this application) that modify geneexpression. It is to be understood that nucleotide sequences, locatedwithin introns, or 3′ of the coding region sequence may also contributeto the regulation of expression of a coding region of interest. Examplesof suitable introns include, but are not limited to, the maize WS6intron, or the maize actin intron. A regulatory element may also includethose elements located downstream (3′) to the site of transcriptioninitiation, or within transcribed regions, or both. In the context ofthe present invention a post-transcriptional regulatory element mayinclude elements that are active following transcription initiation, forexample translational and transcriptional enhancers, translational andtranscriptional repressors, and mRNA stability determinants.

The regulatory elements, or variants or fragments thereof, of thepresent invention may be operatively associated with heterologousregulatory elements or promoters in order to modulate the activity ofthe heterologous regulatory element. Such modulation includes enhancingor repressing transcriptional activity of the heterologous regulatoryelement, modulating post-transcriptional events, or either enhancing orrepressing transcriptional activity of the heterologous regulatoryelement and modulating post-transcriptional events. For example, one ormore regulatory elements, or fragments thereof, of the present inventionmay be operatively associated with constitutive, inducible, or tissuespecific promoters or fragment thereof, to modulate the activity of suchpromoters within desired tissues in plant cells.

The regulatory sequences of the present invention, or variants orfragments thereof, when operably linked to a heterologous nucleotidesequence of interest can drive root-preferred expression of theheterologous nucleotide sequence in the root (or root part) of the plantexpressing this construct. The term “root-preferred,” means thatexpression of the heterologous nucleotide sequence is most abundant inthe root or a root part, including, for example, the root cap, apicalmeristem, protoderm, ground meristem, procambium, endodermis, cortex,vascular cortex, epidermis, and the like. While some level of expressionof the heterologous nucleotide sequence may occur in other plant tissuetypes, expression occurs most abundantly in the root or root part,including primary, lateral and adventitious roots.

A “heterologous nucleotide sequence” is a sequence that is not naturallyoccurring with the promoter sequence of the invention. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.

The isolated promoter sequences of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter region may beutilized and the ability to drive expression of the nucleotide sequenceof interest retained. It is recognized that expression levels of themRNA may be altered in different ways with deletions of portions of thepromoter sequences. The mRNA expression levels may be decreased, oralternatively, expression may be increased as a result of promoterdeletions if, for example, there is a negative regulatory element (for arepressor) that is removed during the truncation process. Generally, atleast about 20 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence.

It is recognized that to increase transcription levels, enhancers may beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like. Someenhancers are also known to alter normal promoter expression patterns,for example, by causing a promoter to be expressed constitutively whenwithout the enhancer, the same promoter is expressed only in onespecific tissue or a few specific tissues.

Modifications of the isolated promoter sequences of the presentinvention can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, a “weak promoter” means a promoter thatdrives expression of a coding sequence at a low level. A “low level” ofexpression is intended to mean expression at levels of about 1/10,000transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts.

It is recognized that the promoters of the invention may be used withtheir native MT2 coding sequences to increase or decrease expression,thereby resulting in a change in phenotype of the transformed plant.This phenotypic change could further affect an increase or decrease inlevels of metal ions in tissues of the transformed plant.

The nucleotide sequences disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant. The MT2 promoter sequences are useful in this aspect whenoperably linked with a heterologous nucleotide sequence whose expressionis to be controlled to achieve a desired phenotypic response. The term“operably linked” means that the transcription or translation of theheterologous nucleotide sequence is under the influence of the promotersequence. In this manner, the nucleotide sequences for the promoters ofthe invention may be provided in expression cassettes along withheterologous nucleotide sequences of interest for expression in theplant of interest, more particularly for expression in the root of theplant.

Such expression cassettes will comprise a transcriptional initiationregion comprising one of the promoter nucleotide sequences of thepresent invention, or variants or fragments thereof, operably linked tothe heterologous nucleotide sequence. Such an expression cassette can beprovided with a plurality of restriction sites for insertion of thenucleotide sequence to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes as well as 3′ termination regions.

The expression cassette can include, in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter, orvariant or fragment thereof, of the invention), a translationalinitiation region, a heterologous nucleotide sequence of interest, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

While it may be preferable to express a heterologous nucleotide sequenceusing the promoters of the invention, the native sequences may beexpressed. Such constructs would change expression levels of the MT2protein in the plant or plant cell. Thus, the phenotype of the plant orplant cell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence beingexpressed, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144;Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al.(1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

The expression cassette comprising the sequences of the presentinvention may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) can be provided on another expression cassette.

Where appropriate, the nucleotide sequences whose expression is to beunder the control of the root-preferred promoter sequence of the presentinvention and any additional nucleotide sequence(s) may be optimized forincreased expression in the transformed plant. That is, these nucleotidesequences can be synthesized using plant preferred codons for improvedexpression. See, for example, Campbell and Gowri (1990) Plant Physiol.92: 1-11 for a discussion of host-preferred codon usage. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989)Nucleic Acids Res. 17: 477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86: 6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allisonet al. (1986) Virology 154: 9-20); MDMV leader (Maize Dwarf MosaicVirus); human immunoglobulin heavy-chain binding protein (BiP) (Macejaket al. (1991) Nature 353: 90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987)Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al.(1989) Molecular Biology of RNA, pages 237-256); and maize chloroticmottle virus leader (MCMV) (Lommel et al. (1991) Virology 81: 382-385).See also Della-Cioppa et al. (1987) Plant Physiology 84: 965-968.Methods known to enhance mRNA stability can also be utilized, forexample, introns, such as the maize Ubiquitin intron (Christensen andQuail (1996) Transgenic Res. 5: 213-218; Christensen et al. (1992) PlantMolecular Biology 18: 675-689) or the maize AdhI intron (Kyozuka et al.(1991) Mol. Gen. Genet. 228: 40-48; Kyozuka et al. (1990) Maydica 35:353-357), and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

Reporter genes or selectable marker genes may be included in theexpression cassettes. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7: 725-737;Goff et al. (1990) EMBO J. 9: 2517-2522; Kain et al. (1995)BioTechniques 19: 650-655; and Chiu et al. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2: 987-992); methotrexate(Herrera Estrella et al. (1983) Nature 303: 209-213; Meijer et al.(1991) Plant Mol. Biol. 16: 807-820); hygromycin (Waldron et al. (1985)Plant Mol. Biol. 5: 103-108; and Zhijian et al. (1995) Plant Science108: 219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7: 171-176);sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136);bromoxynil (Stalker et al. (1988) Science 242: 419-423); glyphosate(Shaw et al. (1986) Science 233: 478-481; and U.S. application Ser. Nos.10/004,357; and 10/427,692); phosphinothricin (DeBlock et al. (1987)EMBO J. 6: 2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (beta-glucuronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5: 387), GFP (green fluorescence protein;Chalfie et al. (1994) Science 263: 802), luciferase (Riggs et al. (1987)Nucleic Acids Res. 15(19): 8115 and Luehrsen et al. (1992) MethodsEnzymol. 216: 397-414) and the maize genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247: 449).

The expression cassette comprising the MT2 promoter of the presentinvention operably linked to a nucleotide sequence of interest can beused to transform any plant. In this manner, genetically modifiedplants, plant cells, plant tissue, seed, root, and the like can beobtained.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4: 320-334), electroporation(Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606),Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055 and Zhao et al., U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissinger etal. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987)Particulate Science and Technology 5: 27-37 (onion); Christou et al.(1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988)Bio/Technology 6: 923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (maize);U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988)Plant Physiol. 91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311: 763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the DNA constructs comprising the promotersequences of the invention can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, viral vector systems and theprecipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, the transcription from theparticle-bound DNA can occur, but the frequency with which its releasedto become integrated into the genome is greatly reduced. Such methodsinclude the use particles coated with polyethylimine (PEI; Sigma#P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5: 209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5: 81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into its genome.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Isolation of Sequence Upstream of the Maize RootMetallothionein 2 (MT2) Gene

The Genome Survey Sequence (GSS) database was used to isolate sequencecorresponding to the promoter of root metallothionein 2 gene (MT2). Afull-length root metallothionein EST sequence was used to search the GSSdatabase for available genomic sequence that overlapped the 5′ end ofthe EST and extended upstream of the EST. Searches of the database wereperformed using BLASTN. Only those sequences displaying greater than 99%identity to the root MT2 gene within the region of overlap wereconsidered significant. The longest available sequence from the searcheswas used to research the GSS database for additional overlappingsequence. This process was repeated until no further overlappingsequence was available. The result of such a series of reiterativesearches was a 1.4 kb region upstream of the root MT2 gene (SEQ ID NO:1).

Example 2 PCR Isolation of the Root Metallothionein 2 Promoter Maize BACclones containing root metallothionein homologs were used as templatesto PCR amplify and isolate about 1.4 kb of sequence corresponding to thesequence obtained through searches of the Genome Survey Sequencedatabase.

PCR primer pairs were used individually with a set of maize BAC clonespreviously known to contain root metallothionein homologs through insilico searching. The PCR primers used in the PCR reactions wereRootmetpro2a (SEQ ID NO:2) and Rootmetpro1b (SEQ ID NO:3).

A BamHI site was added to the 5′ end of Rootmetpro2a primer and a XhoIsite was added to the 3′ end of Rootmetpro1b primer to facilitatesubcloning of the full promoter fragment. The PCR conditions for the PCRamplification reactions were as follows: 95° C. for 15 mins., 25 cyclesof 94° C. for 30 sec, 55° C. for 90 sec, 72° C. for 3 min, and 72° C.for 10 min. A 1.4 kb OCR product was observed with a subset of the BACclones. The resulting 1.4 Kb PCR product was gel purified and clonedinto pCR®4-TOPO® and sequence confirmed, resulting in pBAC11-2.

Example 3 Expression Data Using the Promoter Sequences of the Invention

B73 seeds were placed along one edge of growth paper soaked in asolution of 7% sucrose. An additional piece of growth paper identical insize to the first was also soaked in 7% sucrose and overlaid onto theseeds. The growth paper—seed—growth paper sandwich was subsequentlyjelly rolled with the seed edge at the top of the roll. The roll wasdirectionally placed into a beaker of 7% sucrose solution with the seedsat the top to allow for straight root growth. Seeds were allowed togerminate and develop for 2-3 days in the dark at 27-28° C. Prior tobombardment the outer skin layer of the cotyledon was removed andseedlings were placed in a sterile petri dish (60 mm) on a layer ofWhatman #1 filter paper moistened with 1 mL of H₂O. Two seedlings perplate were arranged in opposite orientations and anchored to the filterpaper with a 0.5% agarose solution. 2-3 cm root tip sections were alsoexcised from seedlings and arranged lengthwise in the plates forbombardment.

DNA/gold particle mixtures were prepared for bombardment in thefollowing method. Sixty mg of 0.6-1.0 micron gold particles werepre-washed with ethanol, rinsed with sterile distilled H₂O, andresuspended in a total of 1 mL of sterile H₂O. 50 μL aliquots of goldparticle suspension were stored in siliconized Eppendorf tubes at roomtemperature. DNA was precipitated onto the surface of the gold particlesby combining, in order, 50 μL aliquot of pre-washed 0.6 μM goldparticles, 5-10 μg of test DNA, 50 μL 2.5 M CaCl₂ and 25 mL of 0.1 Mspermidine. The solution was immediately vortexed for 3 minutes andcentrifuged briefly to pellet the DNA/gold particles. The DNA/gold waswashed once with 500 μL of 100% ethanol and suspended in a final volumeof 50 μL of 100% ethanol. The DNA/gold solution was incubated at −20° C.for at least 60 minutes prior to aliquoting 6 μL of the DNA/gold mixtureonto each Mylar™ macrocarrier. Seedlings prepared as indicated above andexcised root tips were bombarded twice using the PDS-1000/He gun at 1100psi under 27-28 inches of Hg vacuum. The distance between macrocarrierand stopping screen was between 6-8 cm. Plates were incubated in sealedcontainers for 24 hours in the dark at 27-28° C. following bombardment.

After 18-24 hours of incubation the bombarded seedlings and root tipswere assayed for transient GUS expression. Seedlings and excised rootswere immersed in 10-15 mL of assay buffer containing 100 mM NaH₂PO₄—H₂O(pH 7.0), 10 mM EDTA, 0.5 mM K₄Fe(CN)₆—3H₂O, 0.1% Triton X-100 and 2 mM5-bromo-4-chloro-3-indoyl glucuronide. The tissues were incubated in thedark for 24 hours at 37° C. Replacing the GUS staining solution with100% ethanol stopped the assay. GUS expression/staining was visualizedunder a microscope.

Table 1 shows transient bombardment results for the 1.4 kb root MT2promoter:GUS construct, as well as a control ubiquitin promoter:GUSconstruct, in leaf, excised root, and seedling tissue. GUS expressiondriven by the root MT2 promoter was observed in roots and seedlings butnot leaf tissue. GUS expression driven by the ubiquitin controlconstruct was observed in all tissues. TABLE 1 Root MT2 Expression inBombarded Tissues Root MT2 promoter:GUS Ubiquitin promoter:GUS Tissueconstruct expression construct expression leaf ND ++++ root +++ ++++seedling +++ ++++Scoring:+ weak expression levels compared to Ubi:GUS control++ medium expression levels compared to Ubi:GUS control+++ strong expression levels compared to Ubi:GUS control++++ very strong expression levels compared to Ubi:GUS control

Example 4 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a gene of interest operably linked to an MT2 promoterof the invention, or a plasmid comprising the MT2 coding sequences ofthe invention operably linked to a desired promoter (e.g. a ubiquitinpromoter), plus a plasmid containing the selectable marker gene PAT(Wohlleben et al. (1988) Gene 70: 25-37) that confers resistance to theherbicide Bialaphos. Transformation is performed as follows. Mediarecipes follow below.

Preparation of Target Tissue

The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Microdetergent for 20 minutes, and rinsed two times with sterile water. Theimmature embryos are excised and placed embryo axis side down (scutellumside up), 25 embryos per plate, on 560Y medium for 4 hours and thenaligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid vector comprising a gene of interest operably linked to an MTpromoter of the invention is made. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows:

-   -   100 μL prepared tungsten particles in water    -   10 μL (1 μg) DNA in TrisEDTA buffer (1 μg total)    -   100 μL 2.5 M CaCl₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/L Bialaphos,and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to a lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for root-preferred activity of the geneof interest, or for altered metal ion levels.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline(brought to volume with D-1H₂O following adjustment to pH 5.8 with KOH);2.0 g/L Gelrite (added after bringing to volume with D-1H₂O); and 8.5mg/L silver nitrate (added after sterilizing the medium and cooling toroom temperature). Selection medium (560R) comprises 4.0 g/L N6 basalsalts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000XSIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/L Gelrite (added after bringing to volume with D-1H₂O); and0.85 mg/L silver nitrate and 3.0 mg/L bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycinebrought to volume with polished D-1H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15: 473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished D-1H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite (added afterbringing to volume with D-1H₂O); and 1.0 mg/L indoleacetic acid and 3.0mg/L bialaphos (added after sterilizing the medium and cooling to 60°C.).

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycine brought tovolume with polished D-I H₂O), 0.1 g/L myo-inositol, and 40.0 g/Lsucrose (brought to volume with polished D-I H₂O after adjusting pH to5.6); and 6 g/L bacto-agar (added after bringing to volume with polishedD-1H₂O), sterilized and cooled to 60° C.

Example 5 Transformation and Regeneration of Transgenic Plants UsingAgrobacterium Mediated Transformation

For Agrobacterium-mediated transformation of maize with an MT2 promotersequence of the embodiments, the method of Zhao was employed (U.S. Pat.No. 5,981,840, (hereinafter the '840 patent) and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).

Agrobacterium are grown on a master plate of 800 medium and cultured at28° C. in the dark for 3 days, and thereafter stored at 4° C. for up toone month. Working plates of Agrobacterium are grown on 810 mediumplates and incubated in the dark at 28° C. for one to two days.

Briefly, embryos were dissected from fresh, sterilized corn ears andkept in 561Q medium until all required embryos were collected. Embryoswere then contacted with an Agrobacterium suspension prepared from theworking plate, in which the Agrobacterium contained a plasmid comprisingthe promoter sequence of the embodiments. The embryos were co-cultivatedwith the Agrobacterium on 562P plates, with the embryos placed axis downon the plates, as per the '840 patent protocol.

After one week on 562P medium, the embryos were transferred to 5630medium. The embryos were subcultured on fresh 563O medium at 2 weekintervals and incubation was continued under the same conditions. Callusevents began to appear after 6 to 8 weeks on selection.

After the calli had reached the appropriate size, the calli werecultured on regeneration (288W) medium and kept in the dark for 2-3weeks to initiate plant regeneration. Following somatic embryomaturation, well-developed somatic embryos were transferred to mediumfor germination (272V) and transferred to a lighted culture room.Approximately 7-10 days later, developing plantlets were transferred to272V hormone-free medium in tubes for 7-10 days until plantlets werewell established. Plants were then transferred to inserts in flats(equivalent to 2.5″ pot) containing potting soil and grown for 1 week ina growth chamber, subsequently grown an additional 1-2 weeks in thegreenhouse, then transferred to classic 600 pots (1.6 gallon) and grownto maturity. Media used in Agrobacterium-mediated transformation andregeneration of transgenic maize plants:

561Q medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/L thiamine HCl, 68.5g/L sucrose, 36.0 g/L glucose, 1.5 mg/L 2,4-D, and 0.69 g/L L-proline(brought to volume with dI H₂O following adjustment to pH 5.2 with KOH);2.0 g/L Gelrite™ (added after bringing to volume with dI H₂O); and 8.5mg/L silver nitrate (added after sterilizing the medium and cooling toroom temperature).

800 medium comprises 50.0 mL/L stock solution A and 850 mL dI H₂O, andbrought to volume minus 100 mL/L with dI H₂O, after which is added 9.0 gof phytagar. After sterilizing and cooling, 50.0 mL/L stock solution Bis added, along with 5.0 g of glucose and 2.0 mL of a 50 mg/mL stocksolution of spectinomycin. Stock solution A comprises 60.0 g of dibasicK₂HPO₄ and 20.0 g of monobasic sodium phosphate, dissolved in 950 mL ofwater, adjusted to pH 7.0 with KOH, and brought to 1.0 L volume with dIH₂O. Stock solution B comprises 20.0 g NH₄Cl, 6.0 g MgSO₄.7H₂O, 3.0 gpotassium chloride, 0.2 g CaCl₂, and 0.05 g of FeSO₄.7H₂O, all broughtto volume with dI H₂O, sterilized, and cooled.

810 medium comprises 5.0 g yeast extract (Difco), 10.0 g peptone(Difco), 5.0 g NaCl, dissolved in dI H₂O, and brought to volume afteradjusting pH to 6.8. 15.0 g of bacto-agar is then added, the solution issterilized and cooled, and 1.0 mL of a 50 mg/mL stock solution ofspectinomycin is added.

562P medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with dI H₂O followingadjustment to pH 5.8 with KOH); 3.0 g/L Gelrite™ (added after bringingto volume with dI H₂O); and 0.85 mg/L silver nitrate and 1.0 mL of a 100mM stock of acetosyringone (both added after sterilizing the medium andcooling to room temperature).

563O medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, 1.5 mg/L 2,4-D, 0.69 g L-proline, and 0.5 g MES buffer(brought to volume with dI H₂O following adjustment to pH 5.8 with KOH).Then, 6.0 g/L Ultrapure™ agar-agar (EM Science) is added and the mediumis sterilized and cooled. Subsequently, 0.85 mg/L silver nitrate, 3.0 mLof a 1 mg/mL stock of Bialaphos, and 2.0 mL of a 50 mg/mL stock ofcarbenicillin are added.

288 W comprises 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitaminsstock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/Lpyridoxine HCl, and 0.40 g/L Glycine brought to volume with polishedD-1H₂O) (Murashige and Skoog (1962) Physiol. Plant. 15: 473), 100 mg/Lmyo-inositol, 0.5 mg/L zeatin, and 60 g/L sucrose, which is then broughtto volume with polished D-1H₂O after adjusting to pH 5.6. Following, 6.0g/L of Ultrapure™ agar-agar (EM Science) is added and the medium issterilized and cooled. Subsequently, 1.0 mL/L of 0.1 mM abscisic acid;1.0 mg/L indoleacetic acid and 3.0 mg/L Bialaphos are added, along with2.0 mL of a 50 mg/mL stock of carbenicillin.

A recipe for 272V is provided in Example 4.

Example 6 Expression of MT2 in Transgenic Plants

Stable transformed plants were created using Agrobacteriumtransformation protocols as per Example 5, to allow for a more detailedcharacterization of promoter activity.

To begin, leaf and root tissue from regenerated plants growing onnutrient agar stably transformed with an expression cassette containingthe 1347 bp MT2 promoter (SEQ ID NO:1), operably connected to the GUSgene (abbreviated as MT2:GUS), was sampled to test for the presence ofGUS activity. Histochemical analysis showed GUS was expressed inapproximately 60% of the events generated (13 out of 22 events). In thegroup of expressing plants, approximately 62% (8 plants) had expressiononly in roots. No expression was detected in leaves in these 8 plants.For the remaining 5 events, 3 had expression in leaves and roots and 2events had very weak expression in leaves only.

To further characterize the MT2 promoter, fifteen transgenic plants wereforwarded to the greenhouse where they were evaluated under normalgrowing conditions. Seven of the fifteen plants sent were negative forGUS expression while the other 8 plants were from the root-specificevents described above. Leaf and root tissue were sampled from theseplants at the developmental stage, V5 (5 collared leaves). Fourteen outof the 15 plants had expression in nodal roots. Eleven of these plantswere rated as having a level of staining that was comparable to ubi:GUSexpressing plants. Similar results were obtained for lateral roots.Fourteen plants were expressing GUS and 9 of them had levels of GUSstaining comparable to ubi:GUS plants. The ubiquitin promoter isconsidered to be a strong promoter (Christensen et al. (1989) Plant Mol.Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689) and ubi:GUS expressing plants were generated as a positivecontrol for the evaluation of the MT2 events.

No GUS expression was observed in leaves. Abaxial sections of the V5leaf were histochemically stained for GUS and none of the 15 events hadobservable GUS staining. These results correlated well with nativeexpression of the rootmet2 gene using Massively Parallel SignatureSequencing (MPSS) technology (Brenner S. et al. (2000) NatureBiotechnology 18: 630-634, Brenner S. et al. (2000) Proc Natl Acad SciUSA 97: 1665-1670). MPSS involves the generation of 17 base signaturetags from mRNA samples that have been reverse transcribed. The tags aresimultaneously sequenced and assigned to genes or ESTs. The abundance ofthese tags is given a number value that is normalized to parts permillion (PPM) which then allows the tag expression, or tag abundance, tobe compared across different tissues. Thus, the MPSS platform can beused to determine the expression pattern of a particular gene and itsexpression level in different tissues. Expression of the native rootmet2gene in the leaves of maize plants, as determined by MPSS, was 5 PPM (onaverage), which is essentially background level. Expression of the genein roots was approximately 6820 PPM (on average).

The MT2 plants were allowed to mature to R1 stage (silking stage) whereGUS expression in tassels and pollen was also examined. No histochemicalstaining was observed in pollen and only 1 of the 15 events had GUSstaining in the tassel. Again, this correlated well with the MPSS datawhere expression of the rootmet2 gene was 0 PPM in pollen and 23 PPM intassels. The presence of the 35S enhancer altered the expression patternslightly such that GUS was detected in tassels, and in leaves; however,there was no expression in pollen. There also was a slight effect insilks where 3 of 14 plants tested showed low levels of GUS staining.Without the 35S enhancer in the plasmid, none of the MT2 plants (0 outof 14) had silks that stained for GUS. A recheck of the nodal rootsshowed the MT2 promoter was still active. In fact the staining in theroots was as intense, if not more intense, than the staining observed atV5 stage.

Taken together, these data indicate that the MT2 promoter is afunctional genetic element capable of directing transgene expression inmaize plants. The lack of expression in leaves, pollen, tassels, andsilks indicates it is functionally a root-preferred promoter and will beuseful in cases where root-preferred expression is desired.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 1 or a complementthereof; b) a nucleotide sequence comprising the plant promotersequences of the plasmids deposited as Patent Deposit No. NRRL B-30793,or a complement thereof; c) a nucleotide sequence comprising at least 20contiguous nucleotides of the sequence set forth in SEQ ID NO: 1,wherein said sequence initiates transcription in a plant cell; and, d) anucleotide sequence comprising a sequence having at least 95% sequenceidentity to the sequence set forth in SEQ ID NO: 1, wherein saidsequence initiates transcription in the plant cell.
 2. An expressioncassette comprising a nucleotide sequence of claim 1 operably linked toa heterologous nucleotide sequence of interest.
 3. A vector comprisingthe expression cassette of claim
 2. 4. A plant cell comprising theexpression cassette of claim
 2. 5. The plant cell of claim 4, whereinsaid expression cassette is stably integrated into the genome of theplant cell.
 6. The plant cell of claim 4, wherein said plant cell isfrom a monocot.
 7. The plant cell of claim 6, wherein said monocot ismaize.
 8. The plant cell of claim 4, wherein said plant cell is from adicot.
 9. A plant comprising the expression cassette of claim
 2. 10. Theplant of claim 9, wherein said plant is a monocot.
 11. The plant ofclaim 10, wherein said monocot is maize.
 12. The plant of claim 9,wherein said plant is a dicot.
 13. The plant of claim 9, wherein saidexpression cassette is stably incorporated into the genome of the plant.14. A transgenic seed of the plant of claim 13, wherein the seedcomprises the expression cassette.
 15. The plant of claim 9, wherein theheterologous nucleotide sequence of interest comprises a gene productthat confers herbicide, salt, cold, drought, pathogen, or insectresistance.
 16. A method for expressing a nucleotide sequence in a plantor a plant cell, said method comprising introducing into the plant orthe plant cell an expression cassette comprising a promoter operablylinked to a heterologous nucleotide sequence of interest, wherein saidpromoter comprises a nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence comprising the sequence setforth in SEQ ID NO: 1; b) a nucleotide sequence comprising the plantpromoter sequences of the plasmids designated as Patent Deposit No. NRRLB-30793; c) a nucleotide sequence comprising at least 20 contiguousnucleotides of the sequence set forth in SEQ ID NO: 1, wherein saidnucleotide sequence initiates transcription in said plant; and, d) anucleotide sequence comprising a sequence having at least 95% sequenceidentity to the sequence set forth in SEQ ID NO: 1, wherein saidnucleotide sequence initiates transcription in a plant cell.
 17. Themethod of claim 16, wherein the heterologous nucleotide sequence ofinterest comprises a gene product that confers herbicide, salt, cold,drought, pathogen, or insect resistance.
 18. The method of claim 16,wherein said heterologous nucleotide sequence of interest is expressedin a root-preferred manner.
 19. A method for expressing a nucleotidesequence in a root-preferred manner in a plant, said method comprisingintroducing into a plant cell an expression cassette, and regenerating aplant from said plant cell, said plant having stably incorporated intoits genome the expression cassette, said expression cassette comprisinga promoter operably linked to a heterologous nucleotide sequence ofinterest, wherein said promoter comprises a nucleotide sequence selectedfrom the group consisting of: a) a nucleotide sequence comprising thesequence set forth in SEQ ID NO: 1; b) a nucleotide sequence comprisingthe plant promoter sequences of the plasmids deposited as Patent DepositNo. NRRL B-30793; c) a nucleotide sequence comprising at least 20contiguous nucleotides of the sequence set forth in SEQ ID NO: 1,wherein said sequence initiates transcription in a plant root cell; andd) a nucleotide sequence comprising a sequence having at least 95%sequence identity to the sequence set forth in SEQ ID NO: 1, whereinsaid sequence initiates transcription in a plant root cell.
 20. Themethod of claim 19, wherein expression of said heterologous nucleotidesequence of interest alters the phenotype of said plant.